The object of the invention are amino acid sequences (peptides) produced from antibody producing cells, particularly monoclonal antibody producing hybridoma cells, that neutralize the effect of the Clostridium difficile enterotoxin and/or cytotoxin. In addition, there is a description of humanized, monoclonal antibodies for use against the Clostridium difficile toxins, as well as the hypervariable regions of these antibodies. Finally a process for the production and application of these amino acid sequences (peptides) and monoclonal antibodies is shown.
It is known that the introduction of macrolide antibiotics such as Clindamycin leads to severe bowel diseases, which manifest themselves as diarrhoea, proceeding further to occasionally fatal pseudomembranous colitis (PMC). This connection gave the disease the name xe2x80x9cClindamycin associated diarrhoeaxe2x80x9d. We now know that nearly all the antibiotics and cytostatic agents used in medicine can trigger the clinical symptoms of PMC.
PMC is characterised clinically by severe diarrhoea that may lead to death due to heavy electrolyte and liquid losses. Depending on the severity of the symptoms, abdominal pain, bloody diarrhoea, fever and leukocytosis occur. Treatment has hitherto consisted in stopping of the administration of the antibiotic causing the disease, by the administration of Vancomycin, as well as balancing liquid and electrolyte losses.
The aetiological agent of pseudomembranous colitis was for a long time unknown. Only in 1977 could hitherto unknown toxic activity be demonstrated in a stool specimen, inducing a cytotoxic effect on CHO cells (Chinese hamster ovary carcinoma cells). Through further investigations it could finally be proved that the pseudomembranous colitis was caused by Clostridium difficile and its toxins. Clostridium difficile is an obligate anaerobic, gram-positive rod bacteria that builds subterminal oval spores. It is characterised biochemically by being able to ferment monosaccharides such as glucose, N-acetylglucosamine and N-acetylneuraminic acid, but not mannose, xylose or arabinose. Clostridium difficile is also not able to split these monosaccharides off from the side chains of the gastrointestinal mucin as enzymes such as neuraminidase, beta-galactosidase or sialidase are missing. Due to these biochemical shortcomings Clostridium difficile cannot flourish in the stomach of healthy individuals. If the gut flora are disturbed through the administration of antibiotics or cytostatic agents, Clostridium difficile outgrows the gut flora, leading to PMC.
Clostridium difficile produces two major factors of pathogenicity, the enterotoxin (toxin A) and the cytotoxin (toxin B). Their production, purification and properties, together with their use to produce monoclonal antibodies are described in detail in European patents 153 519 and 209 273, U.S. Pat. Nos. 4,879,218 and 5,098,826 as well as international patent application WO 91/18 293.
Antibodies clearly play an important part in protecting against the consequences of infection with Clostridium difficile. Antibody titres against toxin A and toxin B could be established in patients suffering from PMC. After antibiotic treatment and infection with toxinogenic Clostridium difficile strains, hamsters develop the symptoms of PMC and die of it. Prior immunisation of the animals with the above-mentioned toxins protects them from disease. The toxins can be neutralised by antibodies, which are directed against the C-terminal repetitive ligand domains, the central translocation domain or the N-terminal catalytic domain (see reference [1] of the bibliography regarding domain structure). The antibodies directed against the C-terminal repetitive ligand domains hinder the binding of the toxins to the cell receptors, the antibodies directed against the N-terminal catalytic domain block the glucosylation reaction imparted by the toxins, while the antibodies directed against the central translocation domain restrain the translocation of toxins into the cell.
Antibodies neutralising toxin A and/or toxin B thus offer a possible therapy and/or prophylaxis for Clostridium difficile diseases in which they do not eliminate the bacteria but they block the action of the toxins produced. In this way the development of the symptoms of the disease can be hindered by acting on the toxins responsible.
Taking toxin A as an example, the cell line DSM ACC 2322 produces an antibody TTC8, which not only binds to toxin A, but is also able to neutralise its biological action. The binding site of TTC8 in toxin A was identified within the repetitive ligand domain. By binding of the antibodies to the toxins, their interaction with the cell receptors is. hindered. The binding site of mAb TTC8 is within amino acids 2480-2539 of toxin A with the (probable) antigen sequence TINGKKYYF (SEQ ID No. 17). A mAb PCG-4 known from U.S. Pat. No. 4,879,218 binds to a defined protein fragment through the amino acids 2098 to 2141 of toxin A.
The mAb 2CV produced by the cells DSM ACC 2321 also binds to a sequence of the ligand domain, in this case of toxin B. The binding occurs in a protein fragment between aa 2233 and 2366 of toxin B. Both monoclonal antibody PCG-4 and monoclonal antibody TCC8 are mouse antibodies, which are not used in therapy on humans but would be suitable as a diagnostic aid.
The monoclonal antibodies generated in the laboratory of the inventor and their reactivities are indicated below:
All the hitherto generated and described toxin specific monoclonal antibodies were obtained from mice. Therapeutic or prophylactic use of these antibodies in species other than mice (humans or other animals) is not possible since the antibodies are recognised as from a different species, and induce an immune reaction through which they are inactivated. Their adaptation to other species, particularly to humans, has not hitherto been possible since their variable and hypervariable regions were not sequenced (i.e. were not defined).
The objective of the invention was to provide peptides which possess toxin neutralising properties but no longer contain a strongly immunogenic portion, so the peptide is suitable for use in human and veterinary medicine. The neutralisation of the toxin""s effect is based on the binding of the antibodies through the attachment of the antibody variable regions to the respective toxin. This binding is mainly determined through the hypervariable regions (or CDR, complementarity determining regions). It was therefore necessary to identify the regions (variable and hypervariable (CDR)) responsible for binding and neutralising the toxins, and to adapt them in such a way that they no longer triggered an immune reaction. Such peptides can be used both for the therapy of already existing Clostridium difficile diseases and for protection against such diseases in human and veterinary medicine.
The hitherto existing scientific results can be summarised in that, on the one hand, the toxin A of Clostridium difficile is able to trigger the full symptoms of pseudomembranous colitis (PMC) and on the other hand that the monoclonal antibody TTC8 directed against this toxin can neutralise the effect of this toxin during in vivo experiments (in mice). The mAb TTC8 is an antibody of the class IgG 2b. The specific recognition between mAb TTC8 and toxin A of Clostridium difficile is mediated by the hypervariable regions of the heavy and light chains. This means that exactly defined sections of the antibody (in other words, peptides) are responsible for antigen recognition. Knowledge of the hypervariable regions makes it possible to copy these peptides (structures) to antibody sequences of other species, which are then suitable for therapy and prophylaxis of Clostridium difficile diseases, for example in the form of humanised monoclonal antibodies in humans, without having the undesirable side effects of the species-foreign antibodies usually obtained from mice.
It was therefore necessary to determine the nucleotide sequences and the deduced amino acid sequences of the hypervariable, toxin-neutralising monoclonal antibody, so as to be able to use it in the form of peptide sections or humanised antibodies (incorporated into the human antibody gene) for therapy and prophylaxis in humans. A thorough sequencing determination of this type was for example carried out on the cell DSM 2322, which produces the monoclonal antibody TTC8. This antibody neutralises the biological action of enterotoxin A by blocking its ligand domains.
As an example, the hypervariable and variable peptide sequences, which exert the neutralising effect of mAb TTC8, were determined for cell line DSM ACC 2322 with sequences SEQ ID No. 1-12 and/or SEQ ID No. 13-16. Knowledge of the variable regions of neutralising antibodies enables peptides to be produced which block the biological action of Clostridium difficile by binding to its enterotoxin and/or cytotoxin. These peptide sequences, such as SEQ ID No. 14 and/or SEQ ID No. 16 of the sequence protocol, can be used as such or can be incorporated into the immunoglobulin as a therapy for Clostridium difficile diseases. For therapeutic use in humans, human immunoglobulin is used; for use in veterinary medicine, incorporation of the sequences must be carried out into the immunoglobulin gene of the species being treated.
Derivatives of such peptides can be produced, while still preserving their biological activity, by amino acid deletion, insertion, addition or replacement, i.e. by allelic variation. These derivatives, like the original peptides, can inhibit the biological activity of Clostridium difficile through binding to its enterotoxin and/or cytotoxin and can thus be used for the therapy and prophylaxis of Clostridium difficile diseases.
The following named hypervariable regions (CDRs=complementarity determining regions) were determined in mAb TTC8 isolated from hybridoma cell line DSM ACC 2322 deposited in the German collection of microorganisms and cell cultures (DSMZ). They show the following amino acid sequences:
CDRs of the heavy chain:
CDR-1: -Asn-Tyr-Trp-Met-Asn-(SEQ ID No. 2)
CDR-2: -Arg-Ile-Tyr-Pro-Gly-Asp-Gly-Asp-Ala-His-Tyr-Asn-Gly-Lys-Phe-Lys-Gly-(SEQ ID No. 4)
CDR-3: -Gly-Gly-Asn-Tyr-Asp-Asp-Arg-Val-Phe-Asp-Tyr-(SEQ ID No. 6)
CDRs of the light chain:
CDR-4: -Lys-Ala-Ser-Gln-Asn-Val-Gly-Thr-Asn-Val-Ala-(SEQ ID No. 8)
CDR-5: -Ser-Pro-Ser-Tyr-Arg-Tyr-Ser-(SEQ ID No. 10)
CDR-6: -Gln-Gln-Tyr-Asn-Ser-Tyr-Pro-Leu-Thr-(SEQ ID No. 12)
The following procedure was adopted for the determination of these sequences: First the total RNA of the hybridoma cell, which produce the mAb TTC8, was prepared according to a known procedure. The mRNA was then separated in a second step. This was carried out with the help of magnetic polystyrene beads to which the oligo(dT)25 chains are covalently attached.
From the purified mRNA, cDNA was then synthesized and with the help of the polymerase chain reaction (PCR), the VH- and VL-gene of mAb TTC8, which code for the light (VL) or heavy (VH) chain of the TTC8 antibody, were amplified. It was here of decisive importance that primers were selected which showed a suitable restriction site so as to facilitate the subsequent cloning.
The VH- and VL-genes of mAb TTC8 thus produced were subsequently cloned in vector pUC 19 and then sequenced. In this way the nucleotide sequences (SEQ ID No. 13 and 15) and the derived amino acid sequences corresponding to SEQ ID No. 14 and SEQ ID No. 16 were determined.
The hypervariable regions can be identified by means of a comparison with the germ line gene V 102. From the gene of the heavy chain (SEQ ID No. 13) it can be seen that the CDR-1 of the heavy chain contains 5 amino acids and begins at position 30 of the sequence. The CDR-2 of the heavy chain begins at position 49 and contains 17 amino acids. The hypervariable region CDR-3 begins at position 98 and contains 11 amino acids.
Altogether the VH-gene of mAb TTC8 shows 36 point mutations compared to germ line gene V 102. Three of the mutations lie in the binding region of the PCR primer and may therefore be caused by the sequence of the primer. Almost all mutations in the hypervariable regions lead to a change in the amino acid sequence, while nearly half of all mutations in the framework region are inactive.
In the gene for the light chain (SEQ ID No. 15), the hypervariable regions could be established as follows: the CDR-4 begins at position 22 and contains 11 amino acids. The CDR-5 contains 7 amino acids and begins at position 48. The CDR-6 begins at position 87 and contains 9 amino acids. The VL-gene of mAb TTC8 compared to mAb A23 shows only 9 mutations. Of these, 4 lie in the binding region of the PCR primer. Of the five remaining differences in the nucleotide sequence, two mutations are inactive on the protein level. Of the mutations which lead to a change in the amino acid sequence, one lies in the CDR-4, the other two are both in the framework regions 2 and 3.
In a corresponding way, using the hybridoma cell line deposited at the DSMZ (German collection of microorganisms and cell cultures) under number DSM ACC 2321, it is possible to determine the sequences of the hypervariable regions of the monoclonal antibody which recognise the cytotoxin (toxin B) of Clostridium difficile. 
The therapeutic use of a monoclonal antibody derived from mice causes an immune reaction in humans. In order to overcome this problem, an antibody can be adapted (i.e. humanised) to the species to be treated (here humans). There are several procedures for this, which amount to replacing the immunogenic portion of the murine monoclonal antibody with a corresponding portion of a human antibody. They are known from, for example, European patent applications 184 187, 171 496 and 173 494.
The humanised monoclonal antibodies produced from a procedure of this type are considerable more suitable for therapeutic use in humans than the antibodies produced from mice.
These methods for humanising antibodies can also be used for mAb TTC8 and other toxin A and toxin B neutralising monoclonal antibodies. They produce a humanised monoclonal antibody which forms a chimera from the hypervariable regions (e.g. for TTC8: SEQ ID No. 1-12) of the neutralising mAb TTC8 inserted in the framework region of a human immunoglobulin. The latter can be of subtype IgG (preferred for parenteral application) or subtype IgA (preferred for peroral application). As described for the TTC8 antibodies (from the DSM ACC 2322 cell line), the hypervariable regions of other monoclonal antibodies directed against Clostridium difficile can be cloned in a corresponding way, sequenced and used for the production of humanised antibodies.
A further object of the invention are humanised monoclonal antibodies in which the amino acid sequence is modified through allelic variations within the variable and/or constant regions of the light and/or heavy chains of human immunoglobulin, as long as the binding ability to Clostridium difficile toxin A and/or toxin B is maintained.
All the biological xe2x80x9cbuilding blocksxe2x80x9d necessary for the production of monoclonal antibodies according to the invention, such as cell lines, plasmids, promoters, resistance markers, origins of replication, and other fragments of vectors, insofar as they are not deposited as here described, are obtainable on the market or are generally available. If not otherwise stated, they are only used as examples and are not crucial for carrying out the invention, but can be replaced by other suitable biological xe2x80x9cbuilding blocksxe2x80x9d. Bacterial cells are preferably used as hosts for the amplification of the named DNA sequences according to the invention. Examples for this are E. coli or Bacillus spec.
The production of humanised antibodies can be carried out in eukaryote cells such as yeast cells, fungi or, for example, CHO (Chinese hamster ovary cells). The production in plants can be carried out in monocotyledons or dicotyledons.
As an example, the production of antibodies in plants is described below. Production in other organisms is carried out in an analogous way.
The production of specified antibodies in plants is already known from [2] and from international patent application WO 91/06320. For the production of the monoclonal antibody in transgenic plants, the genes for the complete monoclonal antibody or for its recombinant derivatives are cloned, or the gene for a single chain antibody under the control of one or two plant-active promoters in a plant transformation vector is cloned.
The final transformation vector contains all the DNA sequences to be transferred into the plant. These are transferred from the vector into the plant cells. Alternatively the genes for the light and the heavy chains can also be separately incorporated in two plant transformation vectors and separately transferred into plant cells, so as to only later join them together in the final antibody construct. The transformation of plants can be carried out with any suitable method, for example with the help of Agrobacterium tumefaciens or through direct gene transfer or with the help of a gene gun.
The production of antibodies can be directed into predetermined compartments of the cell. By removing the coding sequence for the signal peptide, the antibodies are expressed cytoplasmatically. For high expression of the antibodies, a DNA sequence is connected to the 5xe2x80x2-end of the gene or the naturally occurring one is left, which codes for a signal peptide for transport into the endoplasmic reticulum. In order to obtain specific localisation in a defined cell compartment, it is possible to fuse, onto or into the gene, the DNA sequences which code for the peptide sequences responsible for it. Through integration of a KDEL-sequence it is possible, for example, to secure retention in the endoplasmic reticulum.
The incorporation of the antibody gene into the transgenic gene is analysed and confirmed through suitable restriction digestion of the isolated genomic plant DNA and subsequent Southern hybridisation. Transcription of the gene can be demonstrated using the Northern blot procedure. The biosynthesized antibody is detected using, for example, a specific secondary polyclonal or monoclonal antibody in plant extracts or with a polyclonal or monoclonal antibody directed against the constant region or against a tag-sequence specially introduced for this purpose.
Both monocotyledons, such as barley and wheat, and dicotyledons, such as potatoes, rape, carrots or peas, are suitable as production plants. The expression of the antibody can occur in various organs of the plant, for example in leaves, seeds, tubers or other tissue.
Purification of the antibody expressed in the plant is carried out using, for example, chromatographic methods that are usually also used for purifying antibodies from hybridoma cell lines. Furthermore, recombinant antibodies containing a tag sequence can also be purified by means of specially established affinity chromatography methods such as metal chelation chromatography.
The humanised monoclonal antibodies of the invention can be administered to the human patient for the therapy and prophylaxis of Clostridium difficile caused diseases following known methods. In general the antibodies or antibody fragments of the invention are administered parenterally, or preferably perorally. The direct peroral taking of plants containing the antibodies as raw food can be considered as a special xe2x80x9cgalenic preparationxe2x80x9d. The appropriate dosage of the antibodies of the invention is to be adjusted for the relevant patient and is dependent, for example, on the patient""s body weight, age and disease status. The dosage is set by an experienced medical doctor and usually lies between 0.1 mg/kg and 70 mg/kg, administered once or several times per day over a period of several days.